Reversible phosphorylation of proteins an serine and threonine residues plays a key regulatory role in the transduction of signals that mediate cellular responses to extracellular cues. Whereas protein kinases that catalyze the forward reaction have bee characterized in great detail, much less is known about the protein phosphatases responsible for the reverse reaction. The applicant's laboratory has had a long-standing interest in the study of the protein serine/threonine phosphatase 2A (PP2A) family of enzymes, which is implicated in the control of many aspects of cellular signaling. Substantial progress has been made toward elucidating the structure, function, and regulation of PP2A itself, as well as two novel family members, PP4 and PP6. However, as a group the physiological substrates for these enzymes are poorly defined. Recent published findings from the applicant's laboratory (Science 280:1258, 1998; J. Biol. Chem. 274:687, 1999) indicate that PP2A forms individual macromolecular signaling complexes with several of its protein kinase substrates. Consistent with this discovery, preliminary data presented in this grant application reveal that PP2A also forms a stable multi- protein complex with the IkappaB kinases (IKKs). These kinases are subject to phosphorylation and activation in response to diverse cellular stimuli. The principle substrate of IKK is IkappaB, an inhibitory subunit of the NF-kappaB family of transcription factors. When phosphorylated by IKK, IkappaB is degraded and NF-kappaB is degraded and NF-kappaB is enabled to activate many genes controlling immune and inflammatory responses, cell growth, and apoptosis. Although the precise role of protein phosphatases in this tightly regulated process remains unclear, evidence for the involvement of PP2A family members has recently emerged. The applicant hypothesizes that the IKK-associated phosphatase uncovered in their preliminary studies is a key modulator of NF-kappaB signaling. The following three Specific Aims are proposed to address this central hypothesis: 1) Elucidation of the role PP2A family members play in the cellular regulation of IKK/NF-kappaB signaling; 2) Determination of the holoenzyme form of IKK- associated PP2A and the targeting mechanism of IKK/PP2A interactions; and 3) Identification of specific substrates for the IKK-associated PP2A. These studies will provide fundamental mechanistic insights into the regulation of IKK and NF-kappaB activity by protein phosphatases. Given the key roles that NF-kappaB plays in vivo, the proposed studies may also reveal novel therapeutic targets for diseases affecting immune system function and normal cell growth.